1. Field of the Invention
The present invention relates to the use of “green” or relatively benign solvents such as ethanol, ethanol/water, isopropyl alcohol, isopropyl alcohol/water, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethyl acetate to extract phytosterols from wet corn fiber.
2. Background Art
Cholesterol is an important part of every human's daily diet because it is needed in the body for certain important functions, such as forming nerve cell membranes and aiding the production of hormones. Normally, cholesterol is absorbed in the small intestine and is eventually carried by special lipid and protein complexes called Low Density Lipoproteins (“LDL”). LDL delivers cholesterol to the tissues via the blood and cells that need cholesterol express specific, external LDL receptors. High Density Lipoproteins (“HDL”) collect cholesterol from dying cells and membranes undergoing turnover for recycling. The ratio between LDL and HDL in the bloodstream can be used to predict a patient's health status. If the ratio is above 3.5, then the patient has an elevated LDL or blood cholesterol level. (Biochemistry, 5th Edition, Berg, J. M., et al., eds., W. H. Freeman & Co, New York, N.Y., pp. 726-731 (2002)).
The amount of cholesterol necessary to carry out these functions is, unfortunately, only a tiny portion of the daily cholesterol intake of an average American. When excess cholesterol is ingested or when there is a breakdown in the processing of cholesterol from LDL, it can lead to artherosclerosis or clogged arteries. If an artery to the heart is blocked by an artherosclerotic plaque, it can cause a heart attack. Similar blockage of arteries in the brain can lead to strokes. To lower the risk of a patient having a heart attack or stroke, the blood cholesterol level must be lowered (WO 01/15552). The most direct route to manage blood cholesterol is by lowering dietary intake. There are only a few drugs on the market that inhibit bile recycling or de novo cholesterol biosynthesis (<<http://nhlbisupport.com/chd1/meds.htm>>, National Heart, Lung, and Blood Institute, (2002)).
Phytosterols, or plant fats that include beta-sitosterol and its glucoside beta-sitosterolin, closely resemble the molecule cholesterol (Hicks and Moreau, Food Technology 55(1):63-67 (2001)). Since the mid-1960's, over 5,000 research articles have been published on the positive benefits of phytosterols, including the fact that they interfere with cholesterol absorption. The lowered absorption of cholesterol from the intestines decreases the LDL, lowering plasma cholesterol levels (Nigon, et al., Sang Thrombose Vaisseaux 12(8):483-490 (2000)).
Phytosterols have been found to be present in corn fiber, which is a by-product obtained from “wet-milling” corn (Moreau, et al., Biochemical Society Transactions 28(6):803-806 (2000)). “Wet-milling” is a process by which corn can be separated into its basic components: starch, protein, oil, and fiber (<http://www.com.org/web/process.htm>, Corn Refiners Association, (2002)).
“Corn fiber” is defined in U.S. Pat. No. 5,843,499 as “the product obtained from the wet-milling process, which involves an initial steeping of corn kernels in aqueous sulfur dioxide at an elevated temperature followed by gentle grinding and physical separation of the outer fiber layers from starch, protein and other components” (see also Singh, et al., Cereal Chem. 77(5):665-668 (2000)). For the purpose of this invention, corn fiber will have the meaning just described.
Corn fiber is produced by corn wet-milling at the rate of ˜11% per bushel of corn processed. This means that over 14,000 tons of corn gluten feed and corn oil meal are produced per day, with corn oil meal being a very small fraction (less than 680 tons per day) (<<http://corn.org/web/shipprod.htm>>, Corn Refiners Association, (2003)). Currently, corn fiber is a low value waste stream that, with the addition of protein, can be sold as corn gluten (animal) feed (U.S. Pat. No. 4,038,481). In an effort to upgrade this co-product and harvest phytosterols, a cost-effective strategy was developed. To this end, a solvent system compatible with current corn wet-milling operations is important.
According to Moreau et al. oils containing phytosterols can be extracted from corn fiber using hexanes with the antioxidant BHT present. The Moreau procedures were completed on dried, ground corn fiber at room temperature with agitation. This extraction resulted in mixed oils containing triglycerides (TAG), fatty acid esters of phytosterols (St-FA), free fatty acids (FFA), tocopherols, free phytosterols (St), and ferulic acid esters of phytosterols (St-F). Moreau reported approximately 15% (wt/wt) sterol content with about 7% as St-FA (U.S. Pat. No. 5,843,499).
Moreau's method differs significantly from the present invention in that the extractions described herein may be carried out on either dry corn fiber or wet, unground corn fiber. An advantage to the present invention is that the corn fiber need not be dried or ground. While some reduction of water may be necessary, the lack of need for completely dry corn fiber is an advantage because the energy needed to reduce the fiber from 65% water (typical for wet-milled corn fiber) to 0% water is high. Additionally, grain dust explosions are a huge potential hazard for grain storage and milling operations. This hazard is greatly reduced by not grinding the corn fiber to the mesh grind that the '499 patent suggests. By processing wet corn fiber with larger particle sizes the chance of a grain dust explosion is minimized.
The present invention also differs from the process outlined in the '499 patent because the phytosterols isolated using the method of the present invention are isolated as free sterols, not as a mixture of steryl ferulates, steryl fatty acid esters, and free sterols as seen by Moreau. The present invention provides for both the selective and total extraction of phytosterols based upon the extraction solvent and water content. The saponification conditions applied in the present invention are effective at reducing the oil to two major components: free fatty acids and free sterols.
One goal of the present invention was to optimize a cost-effective method of selectively extracting phytosterols from corn fiber using an environmentally friendly solvent, which is an improvement on the current technology. Green solvents are less toxic than common organic solvents, like hexane (Hanmaoungjai, P., et al., J. Am. Oil Chem. Soc. 78(8):817-821 (2001)). The term “green solvents” in the present disclosure will be considered to comprise water, ethanol, isopropyl alcohol, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethyl acetate and a blend of one or more thereof. These solvents degrade more rapidly in the environment, are less toxic to mammals than many other solvents, and are consistent with the twelve principles of “green chemistry” (<<http://chemistry.org/portal/Chemistry?PID=acsdisplay.html&DOC=education\greenchem\principles.html>>, The American Chemical Society, (2002)).
Ethanol and ethanol/water mixtures are most compatible with current corn wet-milling plants since both ethanol and water are available for use in such plants. The inventors of the present invention have found that pure ethanol extracts have the same yield of corn fiber oil as if extracted by hexane.
Organic solvents do not selectively extract only phytosterols, which is in contrast to the solvents used in the present invention. It is a common known practice to those skilled in the art that extraction of phytosterols can be done by reacting dried, ground corn fiber with organic solvents, such as hexane, chloroform, ether, and methanol. However, these organic solvents extract a mixture of components from the corn fiber, including triglycerides, and therefore the final isolation of the phytosterols from the triglycerides and further purification by crystallization is further complicated.
Purifying a corn lipid extract using crystallization is disclosed in U.S. Pat. No. 6,352,845. The '845 patent discloses an extraction of a corn fiber lipid fraction from wet or dry unground corn fiber using a solvent. After the solvent is removed from the corn fiber lipid fraction, phytosterols and phytosterol esters can be isolated from the corn fiber lipid fraction in a mixture or as individual fractions by crystallization.
WO 00/69280 teaches using ethanol (a green solvent) to extract total oil and zein (protein) from dry-milled corn residue (or corn bran). This is different from the present invention because the starting material for 00/69280 is corn bran, which is a byproduct of dry milling, rather than corn fiber, which is derived from corn wet milling. Corn fiber is a better source of phytosterols than corn bran because corn fiber contains less oil and has a higher phytosterol content. The present invention has optimized the method of selectively extracting the total sterols from corn without any triglycerides or proteins in the final oil product. The wet corn fiber used in the present invention can be taken directly from a wet mill and reacted with a green solvent to start the present disclosed invention.